Supported in part by research grants from the National Institutes of Health (CA73045 and CA23931).
Methods and compositions are presented that use the hepatitis B virus genome, and fragments or extensions thereof, in a baculovirus vector, to develop anti-HBV agents.
Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma.
Although vaccines against HBV exist, vaccination is expensive, not readily available in all parts of the world, and not all individuals develop immunity following vaccination. Therefore, there is a need to develop effective treatments for the millions of people who remain persistently infected, as well as the population who will become infected despite the existence of vaccines. Currently, the only approved therapy for chronic HBV infection is the cytokine interferon-xcex1. Long-term studies on interferon-xcex1 therapy indicate that treatment can lead to the loss of circulating HBV antigens and improved survival rate but only in about 30% of patients receiving treatment. Interferon also must be administered by injection and can have undesirable side effects which limit dosage. Alternative treatment options which are effective alone or in combination with interferon-xcex1 must be explored.
Although much information has accumulated about HBV, knowledge of the virus is by no means complete, therefore prevention and treatment of HBV infections are deficient. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, and the woodchuck hepatitis virus which allows for an in vivo study in an animal model system. The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and the human HBV. For example, avian hepatitis viruses are lacking one of the four HBV open reading frames, and, therefore, do not encode one of the HBV gene products, the X protein. Transcriptional differences between woodchuck hepatitis virus and HBV have been reported. Therefore, animal models may not be reliable for the development of methods to prevent and treat human HBV-related diseases.
Transgenic mice have also been used to study HBV biology. For example, a 1.3 HBV construct was used to generate transgenic mice (Guidotti et al, 1995). The 1.3 HBV transgene was integrated into the mouse genome. These transgenic mouse lineages demonstrate high levels of HBV replication. However, covalently closed circular (CCC) DNA has not been detected as an intermediate in the HBV replication process in these transgenic mice.
Options currently available for studying the molecular mechanisms of HBV replication and the effects of antivirals and cytokines on HBV production within a cell background of human hepatic origin include using stably or transiently transfected cell lines.
Moving closer to predicting human responses to HBV, several HBV expressing cell lines have been established by transfecting viral DNA into liver-derived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. The most widely used cell lines are the HepG2 2.2.15 cell line (2.2.15) (Sells et al., 1987; 1988) derived from the HepG2 hepatoblastoma cell line (Knowles et al., 1980) and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro.
The 2.2.15 cell line which was derived from HepG2 cells and constitutively produces HBV has been used to evaluate in vitro inhibition of HBV replication by various nucleosides. Transient transfection of HepG2 cells have been used to ifs understand various aspects of HBV gene expression and replication at the molecular level. Some studies involve using greater than genome length HBV DNA sequences so that all HBV gene products can be produced. Transcription of linear HBV DNA requires a greater than unit length 3.2-kb HBV genome to produce the 3.5-kb pregenomic message which is required for replication. Others have concentrated on using HBV DNA sequences which encode restricted portions of the coding regions of the genome or enhancer or promoter sequences.
However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. Many HBV expressing cell lines were created using constructs that contain strong heterologous promoters proximal to the HBV genome. A xe2x80x9cheterologousxe2x80x9d promoter is one which is not a natural HBV promoter. The effect those promoters have on HBV transcription and replication is unclear, but effects could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters.
Another reason cell lines may not be predictive of in vivo effects in humans, is that cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not routinely integrated directly into the host genomes but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal, covalently closed circular (CCC) DNA molecules. Although the integration of HBV DNA in human liver has been reported, it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found, it is frequently rearranged and is often transcriptionally silent. Because HBV expressing cell lines contain stably integrated HBV DNA, viral gene expression and replication is continuous; therefore, it is not possible to experimentally control the time or conditions under which these processes are initiated. Stable HBV expressing cell lines contain fixed numbers of integrated HBV genomes and, as such, HBV gene expression and replication levels cannot be regulated and are restricted to the number of integrated copies which each cell line contains. Consequently, it is not possible to study the effects of increasing or decreasing the copy number of integrated HBV genomes without retransfecting the cell line and/or selecting new cell lines.
There is a need, therefore, to develop model systems of HBV infection that more closely mimic actual clinical infections. To accomplish this, a means to initiate HBV replication within human cells in a manner simulating clinical infections is needed. Baculoviruses are a family of large double-stranded DNA viruses which infect and replicate in several types of invertebrate hosts. Baculoviruses have been widely used as insecticides and as agents for protein overexpression in insect cells. Recently, it has been reported that the baculovirus Autographa californica can be used as an effective vector for the transfer of reporter genes into mammalian hepatocytes. (Sanding et al., 1996; Boyce and Bucher, 1996; Hoffman et al., 1995) A requirement for baculovirus-mediated gene expression in hepatocytes appears to be the presence of a promoter which can function in mammalian cells to drive transcription of a target gene. Native baculovirus promoters appear to be ineffective in driving transcription in mammalian cells. There are several advantages of baculovirus-mediated transfer of HBV DNA into HepG2 cells compared to classical transfection procedures: 1) Significantly higher transfection levels ( greater than 10-fold higher) can be obtained. 2) Levels of HBV expression and replication can be precisely and reproducibly controlled simply by altering the multiplicity of infection (moi) with HBV baculovirus. 3) The infection procedure is rapid and requires a minimal manipulation of cultured cells. 4) Cultures of HepG2 cells can be superinfected with additional amounts of baculovirus to extend and/or augment HBV expression levels.
The present invention relates to novel methods and compositions for establishing hepatitis B virus (HBV) gene expression and replication using recombinant HBV baculovirus to deliver the HBV genome to cells in culture.
The invention relates a method for selecting a candidate agent to reduce hepatitis virus expression in human cells, said method comprising:
(a) infecting the cells with a genetic construct comprising at least a part of a baculovirus genome and a greater than a full length hepatitis virus genome, such that it is replication competent, a novel approach;
(b) contacting the infected cells with the candidate agent,
(c) determining whether there is less expression of the hepatitis virus in the contacted cells than in an infected cell that is not contacted with the candidate agent; and
(d) selecting the candidate agent if there is less expression. A suitable candidate agent is 3TC lamivudine. Suitable cell lines are HepG2 or Huh-7. A suitable candidate agent is an antiviral agent or cytokine.
An aspect of the present invention is a hepatic cell infected with a genetic construct comprising at least a part of a baculovirus genome and more than a full length HBV genome such that it is replication competent.
A suitable HBV virus genome that is in the genetic construct is a 1.3-genome length HBV construct which is capable of driving high levels of HBV replication.
An aspect of the invention is a method for determining parameters of HBV infection, said method comprising synchronously initiating HBV replication efficiently in a high, i.e.; about 50-100% percentage of cells of hepatic origin without toxicity to the cells.
An aspect of the invention is a method for transient delivery of high level mammalian gene expression to a cell of hepatic origin using endogenous HBV enhancer and promoter sequences.
Cell lines suitable for practice of the present invention include human cell lines of hepatic origin, e.g. HepG2 and Huh-7. An aspect of the invention is that HBV gene expression is driven exclusively from endogenous promoters, a condition that mimics a natural infection. The methods and compositions of the present invention are alternatives to stable HBV-expressing cell line models and cell lines transiently transfected at low efficiencies by classical procedures. In HBV baculovirus infected cells, HBV transcripts and intracellular and secreted HBV antigens are produced; replication occurs, as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Density-gradient analysis of extracellular HBV DNA indicated that the DNA was contained predominantly in enveloped HBV virions. Covalently closed circular (CCC) DNA is also present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nuclei of infected cells.
An aspect of the invention is that the effects of specific agents (antivirals) cytokines, and so forth) on hepatitis virus gene expression and replication may be tested. This results in a system that can be used to evaluate the efficacy of antivirals for HBV. An HBV recombinant virus system can be used to analyze the effect of an antiviral on HBV replication and on CCC DNA formation. Use of recombinant HBV baculovirus to study HBV replication in cell culture is not limited to the HepG2 cell line but can be extended to the Huh-7 hepatoma cell line and primary human hepatocytes.
Levels of HBV gene expression and replication were achieved in HBV baculovirus-infected HepG2 cells which far exceed levels found in HepG2 2.2.15 cells using previously available methods. In the present invention, HBV baculovirus infection of HepG2 cells lends itself readily to manipulation as follows: 1) HBV expression can be initiated any time relative to seeding of HepG2 cells; 2) levels of HBV replication can be regulated over a wide range simply by changing the baculovirus multiplicity of infection; 3) HBV replication is readily detectable by one day post infection with HBV baculovirus and is detectable at least through day eleven post infection; and (4) the transient nature of the infection can be extended and/or enhanced by superinfecting the cultures. Infection of HepG2 cells by HBV recombinant baculovirus represents a simple to use and highly flexible method for assaying the effects of antivirals and/or cytokines on HBV production, and for understanding HBV replication and pathogenesis at the molecular level.
Abbreviations used herein: HBV: hepatitis B virus; CCC: covalently closed circular; moi: multiplicity of infection; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; LDH: lactate dehydrogenase; HbcAg: hepatitis B core antigen; pfu: plaque forming units.